Serial MRI functional recovery and long
Brain Research Bulletin 61 (2003) 577–585
Serial MRI, functional recovery, and long-term infarct maturation in a
non-human primate model of stroke
J.W.B. Marshall a , R.M. Ridley a,∗ , H.F. Baker a , L.D. Hall b , T.A. Carpenter b,1 , N.I. Wood b,2
a
MRC Comparative Cognition Team, Department of Experimental Psychology, University of Cambridge, Downing Street, Cambridge, CB2 3EB, UK
b Herchel Smith Laboratory for Medicinal Chemistry, University of Cambridge, Robinson Way, Cambridge, CB2 2PZ, UK
Received 17 December 2002; received in revised form 10 June 2003; accepted 1 July 2003
Abstract
We have examined the effects of permanent middle cerebral artery occlusion (pMCAO) in marmoset monkeys over 5 months, using
behavioural and magnetic resonance imaging (MRI) techniques. Three marmosets were trained on behavioural tests before pMCAO. Shortly
after surgery, these marmosets were scanned with T2 -weighted (T2 W) and diffusion-weighted (DW) MRI. Three, 10 and 20 weeks after
surgery, these marmosets were re-tested on the behavioural tasks and had further MRI sessions to monitor lesion development. This was
followed by histological analysis. All these marmosets had a persistent contralesional motor deficit and a spatial neglect which resolved over
the 20 weeks of testing. Percentage infarct volume assessed by MRI on the day of surgery and at 20 weeks matched the percentage infarct
volume measured histologically at 20 weeks. However, the apparent infarct size at 3 weeks was considerably less than that measured by
histological analysis or that measured at the other MRI time points. Additional histological analysis of the brains of two further marmosets
removed 3 weeks after pMCAO found considerable infiltration by lipid filled macrophages into the ischaemic zone which may have caused
an MRI “fogging” effect leading to an apparent reduction in infarct volume.
© 2003 Elsevier Inc. All rights reserved.
Keywords: Marmoset monkeys; Ischaemia; Magnetic resonance imaging; Behaviour
1. Introduction
Magnetic resonance imaging (MRI) has been used in
stroke research to examine the acute oedematous changes
that follow cerebral ischaemia in rodents [7,10,16], in cats
[8,18,23] and to a lesser extent in non-human primates
[3,17,26]. Behavioural studies have also been important in
the development of neuroprotective and restorative drugs for
stroke. However, only a few studies in rodents have combined these approaches [6,24]. The ability both to monitor
infarct development with MRI and to assess the functional
deficits with behavioural techniques in the same animals
could be a powerful tool in stroke research. This is particularly so in primates since their larger brain size provides
∗ Corresponding author. Tel.: +44-1223-339015;
fax: +44-1223-339014.
E-mail address: rmr21@cam.ac.uk (R.M. Ridley).
1 Present address: Wolfson Brain Imaging Centre, Box 65,
Addenbrooke’s Hospital, Hills Road, Cambridge, CB2 2QQ, UK.
2 Present address: Department of Pharmacology, University of
Cambridge, Tennis Court Road, Cambridge, CB2 1QJ, UK.
0361-9230/$ – see front matter © 2003 Elsevier Inc. All rights reserved.
doi:10.1016/S0361-9230(03)00214-4
more detailed MRI scans than can be obtained with rodents,
the brain architecture and, particularly, the territory of the
middle cerebral artery (MCA) is similar in all primates including man, and motor tasks requiring skilled use of the
hand and arm can be used in primates.
We have developed a model of stroke in the marmoset,
a species of New World monkey, to assess whether neuroprotective drugs can reduce long-term functional disability
[13–15]. Unilateral occlusion of the proximal M1 segment
of the MCA produces a large infarct of the frontal, temporal and parietal cortex, the underlying white matter and the
caudate and putamen [12]. Following this occlusion, marmosets have disabilities similar to some of those seen in
stroke patients with damage to the same arterial territory.
These include a transient contralesional spatial neglect, and
a contralesional hemiparesis which, in the marmosets, does
not improve over 10 weeks of testing. The present study was
designed to test the feasibility of combining long-term MRI
and behavioural techniques in a non-human primate species,
to examine the long-term maturation of an experimentally
induced infarct in this species, and to look at functional recovery beyond 10 weeks after an induced stroke.
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2. Materials and methods
2.1. Subjects
Five young adult laboratory-bred, common marmosets
(Callithrix jacchus), aged ∼12 months at the start of the experiment, were used. They were kept within a large colony
and had good visual and auditory interaction with other marmosets. All procedures were carried out in accordance with
United Kingdom Home Office regulations. Three marmosets
(A, M and O) had surgical permanent middle cerebral artery
occlusion (pMCAO), participated in behavioural assessment
and received 4 MRI scans over 20 weeks. Two further marmosets received pMCAO and were assessed histologically
3 weeks later.
2.2. Surgery
The marmosets were anaesthetised with 18 mg/kg Saffan (alphaxolone [9 mg/ml]/alphadolone acetate [3 mg/ml],
0.15 ml/100 g: Glaxo Vet Ltd., Uxbridge, UK) administered
intramuscularly. Under sterile conditions, a large cranial flap
was turned, the dura were opened, and the right MCA was
occluded and bisected as previously described [15]. The site
of occlusion was at the M1 segment of the MCA, 2 mm
medial to the olfactory tract [12]. During surgery, blood
pressure and heart rate were monitored using an Ultrasonic
Doppler Flow Detector (model 811-B; Perimed UK Ltd.,
Bury St Edmunds, UK) with the probe attached to the tail
below an inflatable cuff and pressure gauge. Rectal temperature was also measured, and recordings were taken at 10 min
intervals. The marmosets were transported to the MRI unit
as soon as the surgery was finished while still under anaesthetic.
After the MRI scans, the marmosets were returned to the
marmoset colony and were kept in incubators to maintain
body temperature during recovery from anaesthesia. They
remained in the incubators and were hand-fed and watered
for 2–3 days until they were capable of self-care. Prophylactic antibiotic cover was not required. The marmosets were
injected with analgesic (Finadyne; Schering-Plough Animal
Health Mildenhall UK; 0.01 mg/kg subcutaneously) in the
post-operative period as required.
2.3. Behavioural assessment
The behavioural tasks have been described previously
[11], and therefore only a brief description of each task is
included here. All behavioural testing was performed in a
modified home cage. The marmosets were fed the main part
of their normal daily diet after behavioural testing each day
but food and water deprivation was not required since the rewards used in the tasks (marshmallow pieces) are greatly desired by the marmosets. Before surgery, all marmosets were
familiarised with the tasks after which a formal pre-operative
test was performed. The marmosets were then re-tested 3,
10 and 20 weeks after surgery.
2.3.1. Hill and the Valley staircase tasks
These tasks measure the reaching and grasping ability of
the marmosets. In these tasks the marmosets reached through
vertical slots in a Plexiglas screen, which was attached to the
front of the cage, to retrieve food rewards, placed on the steps
of two staircases on the other side of the transparent screen.
In the Hill task, there were two laterally positioned slots and
the staircases rose towards the centre of the apparatus. The
marmosets used their right arm to reach to the right staircase
and their left arm was used to reach to the left staircase.
In the Valley task, there was one centrally positioned slot
and the staircases rose towards the outside of the apparatus.
The right arm was used to reach to the left staircase and
their left arm was used to reach to the right staircase. Using
these two styles of staircase tasks, a motor impairment in
one arm can be dissociated from a perceptual impairment in
one hemispace (See Fig. 1).
2.3.2. Six tube search task
This task measured the time taken for the marmoset to
search for and retrieve a single reward hidden, on each trial,
in one of the six tubes presented in a horizontal array. The
monkey was free to orientate itself towards the array of tubes
and to use either hand to retrieve the reward. The tube which
contained the reward on each trial was chosen randomly.
This is a test of spatial neglect which assesses the ability of
the marmoset to orient itself within its environment rather
than a test of the spatial boundary of perceptual sensitivity
(see Fig. 2).
2.4. Magnetic resonance imaging
The first MRI session was started 4–6 h after pMCAO
and continued for approximately 6 h. Follow-up MRI sessions were then performed at the end of each post-operative
behavioural testing period: 3, 10 and 20 weeks after surgery.
For imaging, the marmosets were maintained under Saffan
anaesthesia, administered either intravenously at approximately 0.2 ml/h via the tail vein, or intramuscularly, as
required (approximately 0.2 ml/h). Throughout all MRI
sessions, heart rate was monitored by pulse oximetry and
temperature was kept constant with a rectal thermosister
linked to a homeothermic blanket, in which the marmoset
was partially wrapped. The marmoset’s head was inserted
into a 4.35 cm internal diameter 8-strut quadrature birdcage
design, custom-built radio-frequency probe.
MRI was performed with an Oxford Instruments 31 cm
horizontal bore, 2.0 T superconducting magnet, driven by
a Bruker MSL100 console using TOMIKON software.
Pilot images were obtained to ensure correct positioning
of the target images (fast refocused gradient-echo, repetition time (TR) = 100 m s, echo time (TE) = 10 m s, flip
angle = 30◦ , number of excitations per scan (Nex) = 2
with 38 s/scan). The target images covered the entire lesion
(field of view (FoV) = 6 cm, slice thickness = 2 mm).
Diffusion-Weighted spin-echo Images (DW images) (TR =
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Fig. 1. Mean score achieved by marmosets A, M and O on the Hill and Valley staircase tasks, before surgery and 3, 10 and 20 weeks after surgery.
pMCAO was performed in the right hemisphere. The results were analysed by ANOVA followed by post hoc Newman–Keuls analysis, comparing
post-lesion scores with appropriate pre-operative score. ∗∗ P < 0.01.
2500 m s, TE = 54 m s, Nex = 2) were acquired using
a Stejskal-Tanner sequence with a diffusion weighting of
900 s/mm2 . T2 -weighted (T2 W) images were acquired immediately before the DW images, using the same sequence
but without the diffusion gradients. Apparent diffusion coefficient (ADC) maps were obtained from the T2 W and DW
images.
DW imaging detects reduced movement of water
molecules during cytotoxic oedema which results from
stroke and can be seen early after the stroke when the T2 W
image remains normal. The areas of oedema appear bright
and are likely to be the areas with most severe damage and
which are likely to die. T2 W images may detect a number
of pathological features, so ADC maps are calculated from
the MRI scan since only ischaemia results in reduced ADC.
Image analysis was carried out using the in-house software CMRVIEW. Lesion volumes were calculated from the
ADC maps at the post-surgery time point, and from T2 W
images thereafter, by manually drawing around the outline
of the hypointense area of each slice and measuring its area,
as well as that of the two hemispheres. In order to compare
the lesion volume derived from the MRI with that from the
histological sections (see below), the area of lesion on each
MRI slice was determined by subtracting the area of the
hypointense region from the area of the ipsilesional hemisphere, and subtracting this amount from the size of the contralesional hemisphere. Area measurements were then integrated across the anterior–posterior levels to calculate percentage lesion volume.
2.5. Quantitative histological analysis
After the final MRI session, the marmosets were
deeply anaesthetised and were perfused transcardially with
200–300 ml of saline followed by 250–300 ml of 10% formal saline. The brains were removed and immersed in 10%
formal saline. Coronal sections (8 m) were taken at various
anterior–posterior levels through the brains within the areas
of visible infarct. Sections were stained with solochrome
cyanine and counterstained with cresyl violet.
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Fig. 2. Mean time for marmosets A, M and O to find the food reward hidden in one of the six tubes in a horizontal array (A) before pMCAO; (B)
3 weeks after pMCAO; (C) 10 weeks after pMCAO and (D) 20 weeks after pMCAO. The results were analysed by ANOVA followed by post hoc
Newman–Keuls analysis, comparing performance on each tube with the appropriate pre-operative score, ∗∗ P < 0.01.
Solochrome cyanin stained sections, at approximately
2.0 mm intervals through the brain from A 14.5 to P 1.5 of
the stereotaxic atlas [22], were videocaptured with a digital camera connected to a Leica M420 Wild macroscope.
Using a computerised image analysis system (Global Lab
Image, Data Translation Ltd., UK), the area of the remaining ipsilesional hemisphere, excluding areas of peri-infarct
damage and tissue loss, delineated by microscopic analysis
of the sections, was measured. The area of tissue damage
at each level was calculated by subtracting this measure
from the area of the contralesional hemisphere. Area measurements were integrated across the levels to produce
volume measurements for the lesion and for the contralesional hemisphere. The lesion volume as a percentage of
hemispheric volume was then calculated.
paring histology immediately after surgery, at 3, 10 and 20
weeks after pMCAO) would require upwards of six subjects per group, i.e. a total of 24 animals. This highlights
the benefit of MRI in reducing numbers of animals needed
for such timeframe studies. Since a large histological study
was neither feasible nor warranted, it was decided to undertake a more qualitative study to examine the pathological
state of the brain 3 weeks after pMCAO in this species. Two
marmosets therefore received pMCAO and were perfused 3
weeks later, prior to histological examination of the brains.
2.6. Additional histology
Although all marmosets showed impairment on the behavioural tasks, they recovered well from surgery. They were
capable of self-care and of living in a normal cage environment. ‘Automatic’ behaviours such as climbing and jumping were intact but each marmoset exhibited hemiparesis in
the contralesional arm and some disorientation in space.
Once the MRI results had been analysed, we realised that
there was a need for some additional histology from earlier
reference points to understand the progression of pathology.
However, to undertake a quantitative statistical study (com-
3. Results
3.1. Behavioural assessment
J.W.B. Marshall et al. / Brain Research Bulletin 61 (2003) 577–585
3.1.1. Hill and the Valley staircase tasks
The results are shown in Fig. 1. Three factorial analysis
of variance (ANOVA) was used to examine the data using
arm (ipsilesional versus contralesional), staircase (Hill versus Valley) and time (Pre-op, 3, 10 and 20 weeks) as factors.
ANOVA revealed a significant arm×staircase×time interaction [F(3, 24) = 5.736, P < 0.01]. Post hoc Newman–Keuls
analysis was used to compare performance at each time after surgery with pre-operative performance. Pre-operatively
marmosets performed these tasks with ease, successfully retrieving most pieces of reward (maximum score = 15) well
within the time allotted using either their left or right arms.
On all test sessions following pMCAO, the marmosets were
unable to perform the task using their left, contralesional
arm, on both staircase tasks, i.e. reaching into contralesional
and ipsilesional space. This demonstrates a persistent motor
impairment in the contralesional arm.
Although the marmosets were not impaired at reaching
with their right, ipsilesional arm into ipsilesional space on
the Hill task, they were impaired at reaching with their ipsilesional arm into contralesional space on the Valley task at
3 weeks, to a lesser extent at 10 weeks, but not at 20 weeks,
after surgery. This demonstrates a temporary perceptual neglect of contralesional hemispace.
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visible at these time points in T2 W images. T2 W scans are
sensitive to static tissue water, so the absence of hyperintensity in the first scan suggests that there was little acute
vasogenic oedema. Although the lesions were not visible,
it was possible to see in the 7.5 h T2 W images (see Fig.
4 for marmoset O) that the scalp was hyperintense, due to
vasogenic oedema from surgical trauma. Hyperintense regions could be detected in DW images at this time, suggesting reduced diffusion. Restricted water protons experience
relatively less variation in the magnetic gradient and retain
the coherence of their spins, thereby increasing signal intensity. ADC maps most clearly delineated the affected areas at this time and showed non-uniform hypointense areas.
This reduction in ADC in the lesioned hemisphere compared
with the other hemisphere, as mentioned above, suggests decreased water diffusion throughout the affected areas. The
areas affected in the lesioned hemisphere included frontal,
temporal and parietal cortex, as well as the striatum and
sub-cortical white matter in all three marmosets. There was
also some hemispheric swelling, which resulted in a midline
shift towards the contralesional left hemisphere. The poor
lesion definition in T2 W images suggests that this swelling
may not have been caused by vasogenic oedema. However,
the reduced diffusion evident in the ADC maps indicates
that cytotoxic oedema may have been responsible. Fig. 3
3.1.2. Six tube search task
The results are shown in Fig. 2. These data were analysed by two factorial ANOVA with position (tubes 1–6) and
testing phase (Pre-op, 3, 10 and 20 weeks) as factors. There
was a significant position × testing phase interaction effect
[F(5, 15) = 14.610, P < 0.01]. Post hoc Newman–Keuls
analysis was used to compare performance at each time after surgery with pre-operative performance. Pre-operatively,
the marmosets rapidly became accustomed to this test, and
were quick to find rewards wherever they were hidden, although the marmosets were marginally quicker at finding
rewards hidden in the middle tubes of the array than in the
outer ones. When first tested after pMCAO, the marmosets
had severe contralesional hemineglect. Not only did they
fail to find rewards hidden in the tubes on the contralesional
side of the array, they were also impaired at finding rewards
in any tube other than the most ipsilesional tube, to which
they had a marked attraction. By 10 weeks, there was some
recovery, but the marmosets were still significantly slower
at finding rewards hidden in either of the two most contralesional tubes than they had been pre-operatively. The marmosets had recovered 20 weeks after surgery.
3.2. MRI
It took several hours after the stroke had been induced to
complete the surgery, transport the marmosets to the MRI
unit, and adjust the scanner. Therefore, the first time points
at which T2 W and diffusion-weighted (DW) images could
be acquired were ∼4.5 h after pMCAO for marmoset M and
∼7.5 h for marmosets A and O. Lesions were not clearly
Fig. 3. Diffusion-weighted image (DWI) and apparent diffusion coefficient
(ADC) maps from marmosets A and O at 7.5 h after surgery and from
marmoset M 4.5 h after surgery. The vertical line running through the
maps for marmoset M is a zipper artifact. Scale bar = 20 mm.
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shows a DW image and an ADC map from each of the three
marmosets.
Three weeks later, two of the marmosets were re-scanned
(the third marmoset was not scanned at this time because
it was not fit for the prolonged anaesthesia required). There
were noticeable differences between the two hemispheres in
both T2 W and DW images. The loci of damage were similar
to those detected on the day of surgery. The T2 W images
clearly showed non-uniform hyperintense regions. The DW
and ADC images demonstrated heterogeneous lesions, with
patches of hypo- and hyperintensity. In the more posterior
sections, the major change was an increase in volume of the
lateral ventricle. Anterior to stereotaxic level A 4.5, there
was considerable cortical damage extending from the motor
and parietal cortex to the frontal cortex. In all slices, the
ipsilesional lateral ventricle was enlarged, and there was a
small midline distortion towards the ipsilesional hemisphere,
which was shrunken. Figs. 4 and 5 show complete sets of
T2 W images and ADC maps captured from marmoset O on
the day of surgery and 3, 10 and 20 weeks after surgery.
Marmosets A, M and O were scanned 10 and 20 weeks after pMCAO. There was a gradual increase in the infarct size
as shown by T2 W images and ADC maps (see Figs. 4 and
5). These scans show that the ipsilesional lateral ventricle
Fig. 5. Complete set of ADC maps from marmoset O at stereotaxic levels
A 14.5–P1.5 obtained 7.5 h, 3, 10 and 20 weeks after pMCAO. The lesion
can be seen as a bright area in the upper right quadrant of the coronal
sections, particularly at levels A 10.5–A 6.5 at 3 weeks, A 14.5–A 2.5
at 10 weeks and A 14.5–A 0.5 at 20 weeks. The lesion is visible as a
dark area in the same place at 7.5 h after surgery. Scale bar = 20 mm.
was substantially enlarged and the damaged tissue had been
largely replaced by a fluid-filled cyst. There was extensive
damage to all cortical regions, especially the motor cortex,
the dorsal part of the temporal cortex, and some of the parietal cortex. There was damage to the corona radiata and the
external capsule. At both 10 and 20 weeks, the ipsilesional
hemisphere was smaller than the contralesional hemisphere.
3.3. Histology
Marmosets A, M and O had extensive infarcts involving
the whole of the MCA territory including parts of the frontal,
temporal and parietal cortex, the underlying white matter
and the caudate and putamen. The areas of tissue loss and
damage in the histological sections closely matched those
areas seen in the MRI scans. Fig. 6 shows T2 W images
acquired at 20 weeks and the corresponding histological
sections from marmosets A, M and O.
Fig. 4. Complete set of T2 -weighted images from marmoset O at stereotaxic levels A 14.5–P1.5 obtained 7.5 h, 3, 10, and 20 weeks after pMCAO. The lesion can be seen as a bright area in the upper right quadrant
of the coronal sections, particularly at levels A 14.5–A 6.5 at 3 weeks,
A 14.5–A 2.5 at 10 weeks and A 14.5–A0.5 at 20 weeks. The lesion is
not visible 7.5 h after surgery. Scale bar = 20 mm.
3.4. Comparison of MRI and histology
Because marmoset M was not scanned at 3 weeks, comparison of percentage lesion volume as shown by MRI and
by histology was performed only for marmosets A and O.
J.W.B. Marshall et al. / Brain Research Bulletin 61 (2003) 577–585
Fig. 6. T2 W image captured 20 weeks after pMCAO and corresponding
histological sections stained with solochrome cyanin from marmosets A,
M and O.
The average percentage volume of damage (i.e. comparing
the volume of the lesioned hemisphere to the unlesioned
hemisphere between stereotaxic levels A14.5–P1.5) was calculated from ADC maps, T2 W images or histological sections between the stereotaxic levels A14.5–P1.5 (see Fig. 7).
ADC maps constructed on the day of surgery were good
predictors of average percentage lesion volume as measured
histologically after 20 weeks. MR images acquired 3 weeks
after pMCAO were predictive of lesion location but showed
a marked reduction in apparent lesion volume. Ten weeks
after pMCAO, lesion volume had increased and was approximately the same as that estimated on the day of surgery.
There was a small increase in lesion volume by 20 weeks
which closely matched that estimated by histological analysis.
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Fig. 7. Average percentage volume of damage as estimated by MRI at
different time points after surgery or by histology measured at 20 weeks
after surgery. Comparison of the apparent lesion volume at different time
points in the first four columns should be made with the post-mortem
lesion volume as shown in the right hand column. The histogram is the
mean of two marmosets, A and O. Marmoset A is shown as squares, and
marmoset O is shown as circles. Marmoset M could not be scanned at all
time points but scans for marmoset M on the day of surgery and at 20
weeks are shown in Figs. 3 and 6. The ADC map was used to calculate
the lesion size 7.5 h after surgery since the lesion was not visible on T2 W
images at that time. At 3, 10 and 20 weeks the measurements made with
ADC or T2 W were very similar but the T2 W images were clearer and
more detailed, providing more accurate estimates of damage. The lesion
looked smaller in both ADC and T2 W images at 3 weeks than it did at
10 weeks.
The lesion area in the two brains examined histologically
at 3 weeks was partially filled with large round fluid filled
cells, see Fig. 8. In the early development of this model, we
had identified cells with this morphology using RC120 (a
lectin stain) as being lipid filled macrophages. It is likely
that 3 weeks after pMCAO, the area which will develop into
a cyst becomes infiltrated and occupied by macrophages. As
these macrophages clear the necrotic tissue, they become
lipid filled. This would reduce the hyperintensity in the MRI
scans and therefore reduce the apparent lesion size.
3.5. Additional histology
An examination of the two brains perfused 3 weeks after pMCAO revealed areas of tissue degradation in the same
brain areas which comprised the infarct in the marmosets
killed 20 weeks after surgery. In agreement with the size of
the lesion as shown by serial MRI scans, the area of tissue
damage was not as large as that seen in other marmosets
at 20 weeks. However, at this immature stage of lesion development it is difficult to predict how much tissue would
eventually be lost. It is likely that the lesion in these marmosets was similar to that seen in marmosets A, M and O.
Fig. 8. Photomicrograph illustrating the presence of large lipid filled
macrophages within necrotic zone of the infarct. There is no brain tissue
in this section. This area is destined to become a fluid filled cyst when
the lesion is mature. Bar = 50 m.
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4. Discussion
In this study, we have used MRI to monitor the early
oedematous changes that follow an experimentally-induced
stroke in marmosets and have assessed infarct development
and progression of the behavioural deficits over a 5-month
period. We have shown that the motor deficit is a persistent disability whereas the spatial neglect ameliorates with
time. We have reported previously on the deficits seen in the
Staircase and Six Tube Search tasks when the marmosets
were tested up to 10 weeks after pMCAO [11–15]. We have
now extended the test period to 20 weeks and have shown
that while the spatial neglect does ameliorate with time, the
motor disability extends to at least 20 weeks after surgery.
In man, there can be continued recovery of motor function
many months after a stroke, and it is possible that the marmosets would show improvement of the motor disability if
given a longer time to recuperate. Nonetheless, this study
confirms that there is dissociation in time between recovery
from motor disability and recovery from neglect, implying
that they have different neural substrates.
The T2 W images revealed little evidence of the lesion
on the day of surgery, although damage was more obvious
on the DW images. This is consistent with data from studies in rats [5,9] and stroke patients [25]. T2 W imaging has
been used to demonstrate the presence of vasogenic oedema
due to blood–brain barrier breakdown as early as 3.75 h
post-occlusion in the rat photothrombotic model [10], but it
is less sensitive in MCAO models where there is less damage to the blood–brain barrier, and where the time course of
lesion development is slower. The ability of DW protocols to
delineate lesions at early time points may be due to reduced
water diffusion related to cytotoxic oedema [2]. Interruption
of the blood supply leads to an acute shortfall in oxygen
and energy that compromises cell metabolism, causing loss
of cellular osmoregulation. Water then moves from extracellular to intracellular compartments which have lower ADC
values, due to the relative restriction of water protons by intracellular components such as mitochondria and endoplasmic reticulum [19]. Lack of perfusion and hydrostatic pressure in the territory of the occluded MCA may also decrease
the net mobility of water. Although the actual oedema could
be seen most clearly in the DW images, the consequences,
in terms of tissue swelling, could be identified in both the
T2 W and the DW images. There was a midline shift of the
ipsilesional hemisphere towards the contralesional side.
After 3 weeks, the ipsilesional hemisphere had shrunk
with a corresponding increase in the size of the lateral ventricle. Quantitative analysis showed that the lesion volume
was apparently greatly reduced. This phenomenon has been
reported in rats [4,20,24] and was thought to result from an
over-estimate of lesion size in the immediate aftermath of the
ischaemic onset due to tissue oedema and swelling. This is
part of a four-stage pathological process of infarct development that starts with neuronal death, and leads to inflammation, re-organisation and finally, resolution which is thought
to be complete by 30 days in rats [4]. Using MRI, Virley
et al. [24] showed that infarct size in rats remained constant
from 7 to 28 days, by which time lesion development was
considered to be complete. The marmosets in this study were
scanned 3 weeks after their induced stroke, and it might have
been supposed that the infarct would then be near the end of
its development and stable in size. It was therefore surprising that when the marmosets were re-scanned 10 weeks after
pMCAO, the lesion volume had increased and had returned
to that estimated from the ADC maps acquired on the day of
surgery. However, it has been reported in a few human studies that an apparent decrease in T2 W lesion volume occurs
between 1 and 3 weeks after stroke [1,21]. This phenomenon
has been named “MRI fogging”, and although its origins are
not clear, it may be due to (a) magnetically active erythrocytes leaking out of pathologically altered blood vessels 2–3
weeks post-ischaemia and/or (b) the replacement of damaged tissue with scavenging, lipid-laden macrophages and a
decrease in bulk water [21]. Either of these would lead to
masking of the true extent of the lesion when examined using MRI. Our additional histological analysis made 3 weeks
after surgery supports the role that macrophages may play in
this “fogging” effect since there was considerable infiltration
by macrophages into the area of necrotic tissue breakdown.
Once lipid-filled, these macrophages would displace water
reducing the size of damage detected on the T2 W image.
When the number of macrophages have returned to normal,
the true extent of the lesion would again become apparent
on T2 W images. Our MRI results at 10 weeks fit well with
this theory.
At 20 weeks, volumetric analysis showed that there had
been a further apparent increase in infarct volume, although
this increase was not as large as that observed between 3 and
10 weeks. The infarct and ventricle had become confluent
and a large fluid-filled cyst had formed. Following this last
imaging session, the marmosets were perfused and the volume of infarct was calculated from the histological sections.
Percentage measurements were made to allow comparison
between the MR images and the histology as the fixing and
embedding process had caused considerable shrinkage of the
tissue. Using this calculation, the proportion of ischaemic
damage seen in the 20 week images, assessed from either
T2 W images or ADC maps, closely matched that calculated
from the histological sections.
In summary, this study has shown that DW images and
ADC maps obtained on the day of surgery were predictive
of final infarct size, measured histologically 20 weeks after
surgery. The similarity between initial and final lesion volumes suggests that the apparent decrease in lesion size at
3 weeks may be an example of MRI “fogging” caused by
the presence of lipid filled macrophages. The data do indicate, however, that MRI taken shortly after a stroke may
give an accurate prediction of final lesion volume and therefore of the magnitude of the stroke. This information may
be useful for assessing potential neuroprotective treatments
in this model. If marmosets could be scanned quickly after
J.W.B. Marshall et al. / Brain Research Bulletin 61 (2003) 577–585
the onset of stroke before being treated with a putative therapeutic agent, then the MRI data could be used to match the
original damage between different treatment groups, thus
reducing the number of animals needed for each study. Furthermore, an indication from these early scans of the likely
infarct size if left untreated would allow estimation of the
therapeutic value of neuroprotective treatment within each
animal.
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Serial MRI, functional recovery, and long-term infarct maturation in a
non-human primate model of stroke
J.W.B. Marshall a , R.M. Ridley a,∗ , H.F. Baker a , L.D. Hall b , T.A. Carpenter b,1 , N.I. Wood b,2
a
MRC Comparative Cognition Team, Department of Experimental Psychology, University of Cambridge, Downing Street, Cambridge, CB2 3EB, UK
b Herchel Smith Laboratory for Medicinal Chemistry, University of Cambridge, Robinson Way, Cambridge, CB2 2PZ, UK
Received 17 December 2002; received in revised form 10 June 2003; accepted 1 July 2003
Abstract
We have examined the effects of permanent middle cerebral artery occlusion (pMCAO) in marmoset monkeys over 5 months, using
behavioural and magnetic resonance imaging (MRI) techniques. Three marmosets were trained on behavioural tests before pMCAO. Shortly
after surgery, these marmosets were scanned with T2 -weighted (T2 W) and diffusion-weighted (DW) MRI. Three, 10 and 20 weeks after
surgery, these marmosets were re-tested on the behavioural tasks and had further MRI sessions to monitor lesion development. This was
followed by histological analysis. All these marmosets had a persistent contralesional motor deficit and a spatial neglect which resolved over
the 20 weeks of testing. Percentage infarct volume assessed by MRI on the day of surgery and at 20 weeks matched the percentage infarct
volume measured histologically at 20 weeks. However, the apparent infarct size at 3 weeks was considerably less than that measured by
histological analysis or that measured at the other MRI time points. Additional histological analysis of the brains of two further marmosets
removed 3 weeks after pMCAO found considerable infiltration by lipid filled macrophages into the ischaemic zone which may have caused
an MRI “fogging” effect leading to an apparent reduction in infarct volume.
© 2003 Elsevier Inc. All rights reserved.
Keywords: Marmoset monkeys; Ischaemia; Magnetic resonance imaging; Behaviour
1. Introduction
Magnetic resonance imaging (MRI) has been used in
stroke research to examine the acute oedematous changes
that follow cerebral ischaemia in rodents [7,10,16], in cats
[8,18,23] and to a lesser extent in non-human primates
[3,17,26]. Behavioural studies have also been important in
the development of neuroprotective and restorative drugs for
stroke. However, only a few studies in rodents have combined these approaches [6,24]. The ability both to monitor
infarct development with MRI and to assess the functional
deficits with behavioural techniques in the same animals
could be a powerful tool in stroke research. This is particularly so in primates since their larger brain size provides
∗ Corresponding author. Tel.: +44-1223-339015;
fax: +44-1223-339014.
E-mail address: rmr21@cam.ac.uk (R.M. Ridley).
1 Present address: Wolfson Brain Imaging Centre, Box 65,
Addenbrooke’s Hospital, Hills Road, Cambridge, CB2 2QQ, UK.
2 Present address: Department of Pharmacology, University of
Cambridge, Tennis Court Road, Cambridge, CB2 1QJ, UK.
0361-9230/$ – see front matter © 2003 Elsevier Inc. All rights reserved.
doi:10.1016/S0361-9230(03)00214-4
more detailed MRI scans than can be obtained with rodents,
the brain architecture and, particularly, the territory of the
middle cerebral artery (MCA) is similar in all primates including man, and motor tasks requiring skilled use of the
hand and arm can be used in primates.
We have developed a model of stroke in the marmoset,
a species of New World monkey, to assess whether neuroprotective drugs can reduce long-term functional disability
[13–15]. Unilateral occlusion of the proximal M1 segment
of the MCA produces a large infarct of the frontal, temporal and parietal cortex, the underlying white matter and the
caudate and putamen [12]. Following this occlusion, marmosets have disabilities similar to some of those seen in
stroke patients with damage to the same arterial territory.
These include a transient contralesional spatial neglect, and
a contralesional hemiparesis which, in the marmosets, does
not improve over 10 weeks of testing. The present study was
designed to test the feasibility of combining long-term MRI
and behavioural techniques in a non-human primate species,
to examine the long-term maturation of an experimentally
induced infarct in this species, and to look at functional recovery beyond 10 weeks after an induced stroke.
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2. Materials and methods
2.1. Subjects
Five young adult laboratory-bred, common marmosets
(Callithrix jacchus), aged ∼12 months at the start of the experiment, were used. They were kept within a large colony
and had good visual and auditory interaction with other marmosets. All procedures were carried out in accordance with
United Kingdom Home Office regulations. Three marmosets
(A, M and O) had surgical permanent middle cerebral artery
occlusion (pMCAO), participated in behavioural assessment
and received 4 MRI scans over 20 weeks. Two further marmosets received pMCAO and were assessed histologically
3 weeks later.
2.2. Surgery
The marmosets were anaesthetised with 18 mg/kg Saffan (alphaxolone [9 mg/ml]/alphadolone acetate [3 mg/ml],
0.15 ml/100 g: Glaxo Vet Ltd., Uxbridge, UK) administered
intramuscularly. Under sterile conditions, a large cranial flap
was turned, the dura were opened, and the right MCA was
occluded and bisected as previously described [15]. The site
of occlusion was at the M1 segment of the MCA, 2 mm
medial to the olfactory tract [12]. During surgery, blood
pressure and heart rate were monitored using an Ultrasonic
Doppler Flow Detector (model 811-B; Perimed UK Ltd.,
Bury St Edmunds, UK) with the probe attached to the tail
below an inflatable cuff and pressure gauge. Rectal temperature was also measured, and recordings were taken at 10 min
intervals. The marmosets were transported to the MRI unit
as soon as the surgery was finished while still under anaesthetic.
After the MRI scans, the marmosets were returned to the
marmoset colony and were kept in incubators to maintain
body temperature during recovery from anaesthesia. They
remained in the incubators and were hand-fed and watered
for 2–3 days until they were capable of self-care. Prophylactic antibiotic cover was not required. The marmosets were
injected with analgesic (Finadyne; Schering-Plough Animal
Health Mildenhall UK; 0.01 mg/kg subcutaneously) in the
post-operative period as required.
2.3. Behavioural assessment
The behavioural tasks have been described previously
[11], and therefore only a brief description of each task is
included here. All behavioural testing was performed in a
modified home cage. The marmosets were fed the main part
of their normal daily diet after behavioural testing each day
but food and water deprivation was not required since the rewards used in the tasks (marshmallow pieces) are greatly desired by the marmosets. Before surgery, all marmosets were
familiarised with the tasks after which a formal pre-operative
test was performed. The marmosets were then re-tested 3,
10 and 20 weeks after surgery.
2.3.1. Hill and the Valley staircase tasks
These tasks measure the reaching and grasping ability of
the marmosets. In these tasks the marmosets reached through
vertical slots in a Plexiglas screen, which was attached to the
front of the cage, to retrieve food rewards, placed on the steps
of two staircases on the other side of the transparent screen.
In the Hill task, there were two laterally positioned slots and
the staircases rose towards the centre of the apparatus. The
marmosets used their right arm to reach to the right staircase
and their left arm was used to reach to the left staircase.
In the Valley task, there was one centrally positioned slot
and the staircases rose towards the outside of the apparatus.
The right arm was used to reach to the left staircase and
their left arm was used to reach to the right staircase. Using
these two styles of staircase tasks, a motor impairment in
one arm can be dissociated from a perceptual impairment in
one hemispace (See Fig. 1).
2.3.2. Six tube search task
This task measured the time taken for the marmoset to
search for and retrieve a single reward hidden, on each trial,
in one of the six tubes presented in a horizontal array. The
monkey was free to orientate itself towards the array of tubes
and to use either hand to retrieve the reward. The tube which
contained the reward on each trial was chosen randomly.
This is a test of spatial neglect which assesses the ability of
the marmoset to orient itself within its environment rather
than a test of the spatial boundary of perceptual sensitivity
(see Fig. 2).
2.4. Magnetic resonance imaging
The first MRI session was started 4–6 h after pMCAO
and continued for approximately 6 h. Follow-up MRI sessions were then performed at the end of each post-operative
behavioural testing period: 3, 10 and 20 weeks after surgery.
For imaging, the marmosets were maintained under Saffan
anaesthesia, administered either intravenously at approximately 0.2 ml/h via the tail vein, or intramuscularly, as
required (approximately 0.2 ml/h). Throughout all MRI
sessions, heart rate was monitored by pulse oximetry and
temperature was kept constant with a rectal thermosister
linked to a homeothermic blanket, in which the marmoset
was partially wrapped. The marmoset’s head was inserted
into a 4.35 cm internal diameter 8-strut quadrature birdcage
design, custom-built radio-frequency probe.
MRI was performed with an Oxford Instruments 31 cm
horizontal bore, 2.0 T superconducting magnet, driven by
a Bruker MSL100 console using TOMIKON software.
Pilot images were obtained to ensure correct positioning
of the target images (fast refocused gradient-echo, repetition time (TR) = 100 m s, echo time (TE) = 10 m s, flip
angle = 30◦ , number of excitations per scan (Nex) = 2
with 38 s/scan). The target images covered the entire lesion
(field of view (FoV) = 6 cm, slice thickness = 2 mm).
Diffusion-Weighted spin-echo Images (DW images) (TR =
J.W.B. Marshall et al. / Brain Research Bulletin 61 (2003) 577–585
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Fig. 1. Mean score achieved by marmosets A, M and O on the Hill and Valley staircase tasks, before surgery and 3, 10 and 20 weeks after surgery.
pMCAO was performed in the right hemisphere. The results were analysed by ANOVA followed by post hoc Newman–Keuls analysis, comparing
post-lesion scores with appropriate pre-operative score. ∗∗ P < 0.01.
2500 m s, TE = 54 m s, Nex = 2) were acquired using
a Stejskal-Tanner sequence with a diffusion weighting of
900 s/mm2 . T2 -weighted (T2 W) images were acquired immediately before the DW images, using the same sequence
but without the diffusion gradients. Apparent diffusion coefficient (ADC) maps were obtained from the T2 W and DW
images.
DW imaging detects reduced movement of water
molecules during cytotoxic oedema which results from
stroke and can be seen early after the stroke when the T2 W
image remains normal. The areas of oedema appear bright
and are likely to be the areas with most severe damage and
which are likely to die. T2 W images may detect a number
of pathological features, so ADC maps are calculated from
the MRI scan since only ischaemia results in reduced ADC.
Image analysis was carried out using the in-house software CMRVIEW. Lesion volumes were calculated from the
ADC maps at the post-surgery time point, and from T2 W
images thereafter, by manually drawing around the outline
of the hypointense area of each slice and measuring its area,
as well as that of the two hemispheres. In order to compare
the lesion volume derived from the MRI with that from the
histological sections (see below), the area of lesion on each
MRI slice was determined by subtracting the area of the
hypointense region from the area of the ipsilesional hemisphere, and subtracting this amount from the size of the contralesional hemisphere. Area measurements were then integrated across the anterior–posterior levels to calculate percentage lesion volume.
2.5. Quantitative histological analysis
After the final MRI session, the marmosets were
deeply anaesthetised and were perfused transcardially with
200–300 ml of saline followed by 250–300 ml of 10% formal saline. The brains were removed and immersed in 10%
formal saline. Coronal sections (8 m) were taken at various
anterior–posterior levels through the brains within the areas
of visible infarct. Sections were stained with solochrome
cyanine and counterstained with cresyl violet.
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Fig. 2. Mean time for marmosets A, M and O to find the food reward hidden in one of the six tubes in a horizontal array (A) before pMCAO; (B)
3 weeks after pMCAO; (C) 10 weeks after pMCAO and (D) 20 weeks after pMCAO. The results were analysed by ANOVA followed by post hoc
Newman–Keuls analysis, comparing performance on each tube with the appropriate pre-operative score, ∗∗ P < 0.01.
Solochrome cyanin stained sections, at approximately
2.0 mm intervals through the brain from A 14.5 to P 1.5 of
the stereotaxic atlas [22], were videocaptured with a digital camera connected to a Leica M420 Wild macroscope.
Using a computerised image analysis system (Global Lab
Image, Data Translation Ltd., UK), the area of the remaining ipsilesional hemisphere, excluding areas of peri-infarct
damage and tissue loss, delineated by microscopic analysis
of the sections, was measured. The area of tissue damage
at each level was calculated by subtracting this measure
from the area of the contralesional hemisphere. Area measurements were integrated across the levels to produce
volume measurements for the lesion and for the contralesional hemisphere. The lesion volume as a percentage of
hemispheric volume was then calculated.
paring histology immediately after surgery, at 3, 10 and 20
weeks after pMCAO) would require upwards of six subjects per group, i.e. a total of 24 animals. This highlights
the benefit of MRI in reducing numbers of animals needed
for such timeframe studies. Since a large histological study
was neither feasible nor warranted, it was decided to undertake a more qualitative study to examine the pathological
state of the brain 3 weeks after pMCAO in this species. Two
marmosets therefore received pMCAO and were perfused 3
weeks later, prior to histological examination of the brains.
2.6. Additional histology
Although all marmosets showed impairment on the behavioural tasks, they recovered well from surgery. They were
capable of self-care and of living in a normal cage environment. ‘Automatic’ behaviours such as climbing and jumping were intact but each marmoset exhibited hemiparesis in
the contralesional arm and some disorientation in space.
Once the MRI results had been analysed, we realised that
there was a need for some additional histology from earlier
reference points to understand the progression of pathology.
However, to undertake a quantitative statistical study (com-
3. Results
3.1. Behavioural assessment
J.W.B. Marshall et al. / Brain Research Bulletin 61 (2003) 577–585
3.1.1. Hill and the Valley staircase tasks
The results are shown in Fig. 1. Three factorial analysis
of variance (ANOVA) was used to examine the data using
arm (ipsilesional versus contralesional), staircase (Hill versus Valley) and time (Pre-op, 3, 10 and 20 weeks) as factors.
ANOVA revealed a significant arm×staircase×time interaction [F(3, 24) = 5.736, P < 0.01]. Post hoc Newman–Keuls
analysis was used to compare performance at each time after surgery with pre-operative performance. Pre-operatively
marmosets performed these tasks with ease, successfully retrieving most pieces of reward (maximum score = 15) well
within the time allotted using either their left or right arms.
On all test sessions following pMCAO, the marmosets were
unable to perform the task using their left, contralesional
arm, on both staircase tasks, i.e. reaching into contralesional
and ipsilesional space. This demonstrates a persistent motor
impairment in the contralesional arm.
Although the marmosets were not impaired at reaching
with their right, ipsilesional arm into ipsilesional space on
the Hill task, they were impaired at reaching with their ipsilesional arm into contralesional space on the Valley task at
3 weeks, to a lesser extent at 10 weeks, but not at 20 weeks,
after surgery. This demonstrates a temporary perceptual neglect of contralesional hemispace.
581
visible at these time points in T2 W images. T2 W scans are
sensitive to static tissue water, so the absence of hyperintensity in the first scan suggests that there was little acute
vasogenic oedema. Although the lesions were not visible,
it was possible to see in the 7.5 h T2 W images (see Fig.
4 for marmoset O) that the scalp was hyperintense, due to
vasogenic oedema from surgical trauma. Hyperintense regions could be detected in DW images at this time, suggesting reduced diffusion. Restricted water protons experience
relatively less variation in the magnetic gradient and retain
the coherence of their spins, thereby increasing signal intensity. ADC maps most clearly delineated the affected areas at this time and showed non-uniform hypointense areas.
This reduction in ADC in the lesioned hemisphere compared
with the other hemisphere, as mentioned above, suggests decreased water diffusion throughout the affected areas. The
areas affected in the lesioned hemisphere included frontal,
temporal and parietal cortex, as well as the striatum and
sub-cortical white matter in all three marmosets. There was
also some hemispheric swelling, which resulted in a midline
shift towards the contralesional left hemisphere. The poor
lesion definition in T2 W images suggests that this swelling
may not have been caused by vasogenic oedema. However,
the reduced diffusion evident in the ADC maps indicates
that cytotoxic oedema may have been responsible. Fig. 3
3.1.2. Six tube search task
The results are shown in Fig. 2. These data were analysed by two factorial ANOVA with position (tubes 1–6) and
testing phase (Pre-op, 3, 10 and 20 weeks) as factors. There
was a significant position × testing phase interaction effect
[F(5, 15) = 14.610, P < 0.01]. Post hoc Newman–Keuls
analysis was used to compare performance at each time after surgery with pre-operative performance. Pre-operatively,
the marmosets rapidly became accustomed to this test, and
were quick to find rewards wherever they were hidden, although the marmosets were marginally quicker at finding
rewards hidden in the middle tubes of the array than in the
outer ones. When first tested after pMCAO, the marmosets
had severe contralesional hemineglect. Not only did they
fail to find rewards hidden in the tubes on the contralesional
side of the array, they were also impaired at finding rewards
in any tube other than the most ipsilesional tube, to which
they had a marked attraction. By 10 weeks, there was some
recovery, but the marmosets were still significantly slower
at finding rewards hidden in either of the two most contralesional tubes than they had been pre-operatively. The marmosets had recovered 20 weeks after surgery.
3.2. MRI
It took several hours after the stroke had been induced to
complete the surgery, transport the marmosets to the MRI
unit, and adjust the scanner. Therefore, the first time points
at which T2 W and diffusion-weighted (DW) images could
be acquired were ∼4.5 h after pMCAO for marmoset M and
∼7.5 h for marmosets A and O. Lesions were not clearly
Fig. 3. Diffusion-weighted image (DWI) and apparent diffusion coefficient
(ADC) maps from marmosets A and O at 7.5 h after surgery and from
marmoset M 4.5 h after surgery. The vertical line running through the
maps for marmoset M is a zipper artifact. Scale bar = 20 mm.
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shows a DW image and an ADC map from each of the three
marmosets.
Three weeks later, two of the marmosets were re-scanned
(the third marmoset was not scanned at this time because
it was not fit for the prolonged anaesthesia required). There
were noticeable differences between the two hemispheres in
both T2 W and DW images. The loci of damage were similar
to those detected on the day of surgery. The T2 W images
clearly showed non-uniform hyperintense regions. The DW
and ADC images demonstrated heterogeneous lesions, with
patches of hypo- and hyperintensity. In the more posterior
sections, the major change was an increase in volume of the
lateral ventricle. Anterior to stereotaxic level A 4.5, there
was considerable cortical damage extending from the motor
and parietal cortex to the frontal cortex. In all slices, the
ipsilesional lateral ventricle was enlarged, and there was a
small midline distortion towards the ipsilesional hemisphere,
which was shrunken. Figs. 4 and 5 show complete sets of
T2 W images and ADC maps captured from marmoset O on
the day of surgery and 3, 10 and 20 weeks after surgery.
Marmosets A, M and O were scanned 10 and 20 weeks after pMCAO. There was a gradual increase in the infarct size
as shown by T2 W images and ADC maps (see Figs. 4 and
5). These scans show that the ipsilesional lateral ventricle
Fig. 5. Complete set of ADC maps from marmoset O at stereotaxic levels
A 14.5–P1.5 obtained 7.5 h, 3, 10 and 20 weeks after pMCAO. The lesion
can be seen as a bright area in the upper right quadrant of the coronal
sections, particularly at levels A 10.5–A 6.5 at 3 weeks, A 14.5–A 2.5
at 10 weeks and A 14.5–A 0.5 at 20 weeks. The lesion is visible as a
dark area in the same place at 7.5 h after surgery. Scale bar = 20 mm.
was substantially enlarged and the damaged tissue had been
largely replaced by a fluid-filled cyst. There was extensive
damage to all cortical regions, especially the motor cortex,
the dorsal part of the temporal cortex, and some of the parietal cortex. There was damage to the corona radiata and the
external capsule. At both 10 and 20 weeks, the ipsilesional
hemisphere was smaller than the contralesional hemisphere.
3.3. Histology
Marmosets A, M and O had extensive infarcts involving
the whole of the MCA territory including parts of the frontal,
temporal and parietal cortex, the underlying white matter
and the caudate and putamen. The areas of tissue loss and
damage in the histological sections closely matched those
areas seen in the MRI scans. Fig. 6 shows T2 W images
acquired at 20 weeks and the corresponding histological
sections from marmosets A, M and O.
Fig. 4. Complete set of T2 -weighted images from marmoset O at stereotaxic levels A 14.5–P1.5 obtained 7.5 h, 3, 10, and 20 weeks after pMCAO. The lesion can be seen as a bright area in the upper right quadrant
of the coronal sections, particularly at levels A 14.5–A 6.5 at 3 weeks,
A 14.5–A 2.5 at 10 weeks and A 14.5–A0.5 at 20 weeks. The lesion is
not visible 7.5 h after surgery. Scale bar = 20 mm.
3.4. Comparison of MRI and histology
Because marmoset M was not scanned at 3 weeks, comparison of percentage lesion volume as shown by MRI and
by histology was performed only for marmosets A and O.
J.W.B. Marshall et al. / Brain Research Bulletin 61 (2003) 577–585
Fig. 6. T2 W image captured 20 weeks after pMCAO and corresponding
histological sections stained with solochrome cyanin from marmosets A,
M and O.
The average percentage volume of damage (i.e. comparing
the volume of the lesioned hemisphere to the unlesioned
hemisphere between stereotaxic levels A14.5–P1.5) was calculated from ADC maps, T2 W images or histological sections between the stereotaxic levels A14.5–P1.5 (see Fig. 7).
ADC maps constructed on the day of surgery were good
predictors of average percentage lesion volume as measured
histologically after 20 weeks. MR images acquired 3 weeks
after pMCAO were predictive of lesion location but showed
a marked reduction in apparent lesion volume. Ten weeks
after pMCAO, lesion volume had increased and was approximately the same as that estimated on the day of surgery.
There was a small increase in lesion volume by 20 weeks
which closely matched that estimated by histological analysis.
583
Fig. 7. Average percentage volume of damage as estimated by MRI at
different time points after surgery or by histology measured at 20 weeks
after surgery. Comparison of the apparent lesion volume at different time
points in the first four columns should be made with the post-mortem
lesion volume as shown in the right hand column. The histogram is the
mean of two marmosets, A and O. Marmoset A is shown as squares, and
marmoset O is shown as circles. Marmoset M could not be scanned at all
time points but scans for marmoset M on the day of surgery and at 20
weeks are shown in Figs. 3 and 6. The ADC map was used to calculate
the lesion size 7.5 h after surgery since the lesion was not visible on T2 W
images at that time. At 3, 10 and 20 weeks the measurements made with
ADC or T2 W were very similar but the T2 W images were clearer and
more detailed, providing more accurate estimates of damage. The lesion
looked smaller in both ADC and T2 W images at 3 weeks than it did at
10 weeks.
The lesion area in the two brains examined histologically
at 3 weeks was partially filled with large round fluid filled
cells, see Fig. 8. In the early development of this model, we
had identified cells with this morphology using RC120 (a
lectin stain) as being lipid filled macrophages. It is likely
that 3 weeks after pMCAO, the area which will develop into
a cyst becomes infiltrated and occupied by macrophages. As
these macrophages clear the necrotic tissue, they become
lipid filled. This would reduce the hyperintensity in the MRI
scans and therefore reduce the apparent lesion size.
3.5. Additional histology
An examination of the two brains perfused 3 weeks after pMCAO revealed areas of tissue degradation in the same
brain areas which comprised the infarct in the marmosets
killed 20 weeks after surgery. In agreement with the size of
the lesion as shown by serial MRI scans, the area of tissue
damage was not as large as that seen in other marmosets
at 20 weeks. However, at this immature stage of lesion development it is difficult to predict how much tissue would
eventually be lost. It is likely that the lesion in these marmosets was similar to that seen in marmosets A, M and O.
Fig. 8. Photomicrograph illustrating the presence of large lipid filled
macrophages within necrotic zone of the infarct. There is no brain tissue
in this section. This area is destined to become a fluid filled cyst when
the lesion is mature. Bar = 50 m.
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J.W.B. Marshall et al. / Brain Research Bulletin 61 (2003) 577–585
4. Discussion
In this study, we have used MRI to monitor the early
oedematous changes that follow an experimentally-induced
stroke in marmosets and have assessed infarct development
and progression of the behavioural deficits over a 5-month
period. We have shown that the motor deficit is a persistent disability whereas the spatial neglect ameliorates with
time. We have reported previously on the deficits seen in the
Staircase and Six Tube Search tasks when the marmosets
were tested up to 10 weeks after pMCAO [11–15]. We have
now extended the test period to 20 weeks and have shown
that while the spatial neglect does ameliorate with time, the
motor disability extends to at least 20 weeks after surgery.
In man, there can be continued recovery of motor function
many months after a stroke, and it is possible that the marmosets would show improvement of the motor disability if
given a longer time to recuperate. Nonetheless, this study
confirms that there is dissociation in time between recovery
from motor disability and recovery from neglect, implying
that they have different neural substrates.
The T2 W images revealed little evidence of the lesion
on the day of surgery, although damage was more obvious
on the DW images. This is consistent with data from studies in rats [5,9] and stroke patients [25]. T2 W imaging has
been used to demonstrate the presence of vasogenic oedema
due to blood–brain barrier breakdown as early as 3.75 h
post-occlusion in the rat photothrombotic model [10], but it
is less sensitive in MCAO models where there is less damage to the blood–brain barrier, and where the time course of
lesion development is slower. The ability of DW protocols to
delineate lesions at early time points may be due to reduced
water diffusion related to cytotoxic oedema [2]. Interruption
of the blood supply leads to an acute shortfall in oxygen
and energy that compromises cell metabolism, causing loss
of cellular osmoregulation. Water then moves from extracellular to intracellular compartments which have lower ADC
values, due to the relative restriction of water protons by intracellular components such as mitochondria and endoplasmic reticulum [19]. Lack of perfusion and hydrostatic pressure in the territory of the occluded MCA may also decrease
the net mobility of water. Although the actual oedema could
be seen most clearly in the DW images, the consequences,
in terms of tissue swelling, could be identified in both the
T2 W and the DW images. There was a midline shift of the
ipsilesional hemisphere towards the contralesional side.
After 3 weeks, the ipsilesional hemisphere had shrunk
with a corresponding increase in the size of the lateral ventricle. Quantitative analysis showed that the lesion volume
was apparently greatly reduced. This phenomenon has been
reported in rats [4,20,24] and was thought to result from an
over-estimate of lesion size in the immediate aftermath of the
ischaemic onset due to tissue oedema and swelling. This is
part of a four-stage pathological process of infarct development that starts with neuronal death, and leads to inflammation, re-organisation and finally, resolution which is thought
to be complete by 30 days in rats [4]. Using MRI, Virley
et al. [24] showed that infarct size in rats remained constant
from 7 to 28 days, by which time lesion development was
considered to be complete. The marmosets in this study were
scanned 3 weeks after their induced stroke, and it might have
been supposed that the infarct would then be near the end of
its development and stable in size. It was therefore surprising that when the marmosets were re-scanned 10 weeks after
pMCAO, the lesion volume had increased and had returned
to that estimated from the ADC maps acquired on the day of
surgery. However, it has been reported in a few human studies that an apparent decrease in T2 W lesion volume occurs
between 1 and 3 weeks after stroke [1,21]. This phenomenon
has been named “MRI fogging”, and although its origins are
not clear, it may be due to (a) magnetically active erythrocytes leaking out of pathologically altered blood vessels 2–3
weeks post-ischaemia and/or (b) the replacement of damaged tissue with scavenging, lipid-laden macrophages and a
decrease in bulk water [21]. Either of these would lead to
masking of the true extent of the lesion when examined using MRI. Our additional histological analysis made 3 weeks
after surgery supports the role that macrophages may play in
this “fogging” effect since there was considerable infiltration
by macrophages into the area of necrotic tissue breakdown.
Once lipid-filled, these macrophages would displace water
reducing the size of damage detected on the T2 W image.
When the number of macrophages have returned to normal,
the true extent of the lesion would again become apparent
on T2 W images. Our MRI results at 10 weeks fit well with
this theory.
At 20 weeks, volumetric analysis showed that there had
been a further apparent increase in infarct volume, although
this increase was not as large as that observed between 3 and
10 weeks. The infarct and ventricle had become confluent
and a large fluid-filled cyst had formed. Following this last
imaging session, the marmosets were perfused and the volume of infarct was calculated from the histological sections.
Percentage measurements were made to allow comparison
between the MR images and the histology as the fixing and
embedding process had caused considerable shrinkage of the
tissue. Using this calculation, the proportion of ischaemic
damage seen in the 20 week images, assessed from either
T2 W images or ADC maps, closely matched that calculated
from the histological sections.
In summary, this study has shown that DW images and
ADC maps obtained on the day of surgery were predictive
of final infarct size, measured histologically 20 weeks after
surgery. The similarity between initial and final lesion volumes suggests that the apparent decrease in lesion size at
3 weeks may be an example of MRI “fogging” caused by
the presence of lipid filled macrophages. The data do indicate, however, that MRI taken shortly after a stroke may
give an accurate prediction of final lesion volume and therefore of the magnitude of the stroke. This information may
be useful for assessing potential neuroprotective treatments
in this model. If marmosets could be scanned quickly after
J.W.B. Marshall et al. / Brain Research Bulletin 61 (2003) 577–585
the onset of stroke before being treated with a putative therapeutic agent, then the MRI data could be used to match the
original damage between different treatment groups, thus
reducing the number of animals needed for each study. Furthermore, an indication from these early scans of the likely
infarct size if left untreated would allow estimation of the
therapeutic value of neuroprotective treatment within each
animal.
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